Chem. Res. Toxicol. 2008, 21, 1011–1016
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Communication Molecular Structure and Dynamic Properties of a Sulfonamide Derivative of Glutathione That Is Produced Under Conditions of Oxidative Stress by Hypochlorous Acid D. Tim Harwood,† Susan L. Nimmo,‡ Anthony J. Kettle,† Christine C. Winterbourn,† and Michael T. Ashby*,‡ Free Radical Research Group, Department of Pathology, UniVersity of Otago, Christchurch, New Zealand, and Department of Chemistry and Biochemistry, UniVersity of Oklahoma, Norman, Oklahoma 73019 ReceiVed February 7, 2008
Reduced glutathione (GSH) is a cornerstone of the antioxidant stratagem for eukaryotes and some prokaryotes. Hypochlorous acid (HOCl), which is produced by neutrophilic myeloperoxidase, reacts rapidly with excess GSH to yield mainly oxidized glutathione (GSSG). GSSG can be further oxidized to give first N-chloro derivatives and, later, higher oxidation states at the S centers. Under certain conditions, another major species that is observed during the oxidation of GSH by HOCl (and a minor species for other oxidants) exhibits a molecular mass that is 30 mass units heavier than GSH. This GSH+2O-2H species, which has been employed as a biomarker for oxidative stress, has been previously proposed to be a sulfonamide. Employing NMR spectroscopy and mass spectrometry, we demonstrate that the GSH+2O-2H species is indeed a nine-membered cyclic sulfonamide. Alternative formulations, including six-membered 1,2,5-oxathiazine heterocycles, have been ruled out. Remarkably, the sulfonamide exists as a 2:1 equilibrium mixture of two diastereomers. Isotope tracer studies have demonstrated that it is the Glu CR center that has undergone racemization. It is proposed that the racemization takes place via an acyclic imine-sulfinic acid intermediate. The glutathione sulfonamides are stable products of GSH that have been detected in physiological systems. Elucidation of the structures of the glutathione sulfonamides provides further impetus to explore their potential as biomarkers of hypochlorous acid formation. Introduction
Scheme 1. Oxidation Derivatives of GSH
The tripeptide glutathione (GSH) is the principal intracellular nonprotein thiol in eukaryotes (1) and some prokaryotes (2, 3), and as such, it serves to maintain intracellular redox states. GSH acts as a substrate for various enzymes involved in redox regulation (4), and it can also be oxidized directly by various reactive oxygen species (ROS) (5). There is mounting evidence that two-electron oxidants, such as the neutrophil-derived hypochlorous acid (HOCl), may play significant roles in inflammatory diseases (6). Mechanistic investigations of the rates of reaction of HOCl with physiological constituents that have been studied thus far reveal that the sulfur-containing compounds cysteine (CySH) (7–11), methionine (Met) (9, 10), and thiocyanate (SCN-) (12, 13) are most reactive. Because of its possible connection to inflammatory diseases, there is considerable interest in identifying biomarkers for oxidative stress by HOCl, and GSH-derived products are potential sources for these biomarkers. A number of products of the reaction of GSH and HOCl have been identified, including those of Scheme 1. Of course, oxidized derivatives of GSH that are reversibly reduced or that are of transitory nature cannot * To whom correspondence should be addressed. Tel: 405-325-2924. Fax: 405-325-6111. E-mail:
[email protected]. † University of Otago Christchurch. ‡ University of Oklahoma.
serve as useful biomarkers. For the derivatives of Scheme 1, only GSSG has been generally known to be reversibly reduced (e.g., by GSH reductase), but with the discovery of sulfinate
10.1021/tx800050n CCC: $40.75 2008 American Chemical Society Published on Web 05/01/2008
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Scheme 2. Alternative Structure for the GSH+2O-2H Species
Communications
structure of GSA using NMR. Complementary MS data are also presented. The proposed sulfonamide structure has been established by the present study, but we surprisingly find that it exists in two diastereomeric forms that interconvert vis-a`-vis epimerization of the Glu CR stereocenter.
Materials and Methods
(14) and sulfonate (15) reductases, it is conceivable that GSO2H and GSO3H are also reduced in vivo. The ester derivatives of GSH are also unstable, and they are known to spontaneously react to give mixtures of GSH, GSSG, GSO2H, and GSO3H, depending on the reaction conditions. In addition to the sulfur center, the amine moiety of the Glu residue of GSH derivatives can also be oxidized by hypohalous acids to initially produce the haloamines [for example, GSSG gives N-chloro and N,N′dichloro derivatives (16)]. However, the haloamines are also transitory reactive intermediates (16), and as such, they are not useful biomarkers. In addition to the fully characterized GSH derivatives of Scheme 1, two novel structurally uncharacterized oxidation products have been identified that exhibit nominal molecular masses that are two mass units lighter (GSH-2H) and 30 mass units heavier (GSH+2O-2H) than GSH (17). The latter derivative is the focus of this paper. On the basis of the mass of the parent peak and the fact that the GSH+2O-2H species did not react with o-phthalaldehyde or ninhydrin (suggesting that it did not contain an amino group), it has been proposed that the GSH+2O-2H species contains a covalent sulfonamide bond between the cysteinyl sulfur and the amino group of the γ-glutamyl residue of GSH (GSA). GSA is produced by the MPO system (18), it has been detected in neutrophils and endothelial cells that have been treated with HOCl (19, 20), and it has been observed in PMA-stimulated neutrophils (21). Apart from the original characterization of GSA (17), no structural studies have been performed. Because the proposed structure contains an unusual nine-membered (mesocyclic) ring (22), strained aliphatic sulfonamide groups with reactive neighboring groups are generally unstable in an aqueous environment (23–27), and alternative structures are consistent with the observed mass (Scheme 2), we sought to confirm the
Reagents. All chemicals were ACS certified grade or better. GSH was from Sigma-Aldrich (St. Louis, MO). Acetonitrile was from Mallinckrodt Baker (Paris, KY). NaOCl was from Sarah Lee (Auckland, NZ), and its concentration was determined spectrophotometrically (pH 12, 292 ) 350 M-1 cm-1). Deuterium oxide (99.9%) and labeled GSH ([glycine 1,213 C2,15N]GSH) were from Cambridge Isotope Laboratories (Andover, MA). Water was doubly distilled in glass. The buffer solutions were prepared from K3PO4, and the pH/pD was adjusted with NaOH or NaOD (prepared by the careful addition of Na sand to D2O under an atmosphere of Ar. Caution: Hydrogen gas evolves and can be ignited by the hot Na if O2 is not excluded. pH/pD Measurements. The [OH-] for the unbuffered solutions was determined by acid-base titration against standardized HCl solutions. The [H+] of the buffered solutions was determined with an Orion Ion Analyzer EA920 using an Ag/ AgCl combination pH electrode. pD measurements in D2O were made using the same pH electrode by adding 0.4 units to the measurement (28). Synthesis of GSA. GSA was synthesized by the reaction of GSH with an equimolar amount of HOCl. HOCl (5 mL of a 100 mM solution in 100 mM phosphate buffer, pH 7.4) was added to 5 mL of a stirred solution of 100 mM GSH in water at room temperature. The equimolar ratio of thiol and oxidant was chosen because it gave an acceptable yield of GSA without forming excessive amounts of GSO3H or GSO2SG. More GSA could be formed with the addition of greater amounts of HOCl, but the reaction mixture became discolored and led to an impure preparation of the sulfonamide. GSA was formed immediately upon mixing, and it could be subsequently separated on a Phenomenex Jupiter semipreparative C18 column (4.6 mm × 250 mm) using an isocratic elution (50 mM formic acid, pH 2.4) with UV detection at 222 nm. GSA eluted at 8 min, baseline resolved from the other components of the reaction mixture. The GSA was manually collected and then freeze-dried to give a fluffy off-white powder that was stored at -20 °C until use. Importantly, lower yields of GSA were formed when turbulent mixing (e.g., stopped-flow) conditions were applied. To generate GSA containing an isotopically labeled glycine residue, commercially available [glycine 1,2-13C2,15N]GSH was oxidized with an equal volume of an equimolar amount of HOCl. The conditions for isolation were the same as those that were used for unlabeled GSA. Once formed, GSA was stable in water or 10 mM phosphate buffer (pH 7.4) for at least 6 months at -20 °C. NMR Studies. 1H (499.883 MHz), 2H (76.728 MHz), 13C (125.708 MHz), and 15N (50.657 MHz) NMR spectra were recorded with a Varian VNMRS 500 spectrometer at the indicated temperatures ((0.5) °C using the 1D presaturation gradient-selected COSY, gradient-selected absolute value HMBC, gradient-selected 1H-13C HSQC, and 1H-15N HSQC gradient sensitivity enhanced for 15N pulse sequences as supplied by the Varian Biopack software. The 1H chemical shifts (ppm) were referenced to sodium 2,2-dimethyl-2-silapentane-5-sulfonate
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Table 1. 1H NMR Chemical Shifts (ppm) of Reduced GSH, Oxidized Glutathione (GSSG), Glutathione Sulfonate (GSO3-), and the Major and Minor Diastereomers of Gluathione Sulfonamide (GSA) at pH 7.4, 22 °C, and 500 MHz Gly R-Ha Gly R-Hb Gly NH Cys R-H Cys β-Ha Cys β-Hb Cys NH Glu R-H Glu β-Ha Glu β-Hb Glu γ-Ha Glu γ-Hb
GSH
GSSG
GSO3-
GSA (major)
GSA (minor)
3.78 3.79 8.29 4.58 2.94 2.99 8.51 3.79 2.17 2.17 2.54 2.56
3.78 3.78 8.33 4.80 2.97 3.31 8.61 3.78 2.16 2.16 2.51 2.57
3.73 3.76 8.29 4.84 3.28 3.47 8.51 3.77 2.14 2.20 2.52 2.56
3.75 3.87 8.45 4.75a 3.45 3.58 9.68b 4.53 2.17 2.61 3.03 3.05
3.76 3.88 8.67 4.73a 3.32 3.61 9.67b 4.51 2.20 2.62 3.08 3.14
Under the water peak. b Measured at 2 °C.
a
Table 2. 1H NMR Coupling Constants (Hz) of Reduced GSH, GSSG, GSO3-, and the Major and Minor Diastereomers of GSA at pH 7.4, 22 °C, and 500 MHz GSH
GSSG
GSO3-
3
JGlyR-Ha,GlyR-Hb 3 JCysR-H,Cysβ-Ha 3 JCysR-H,Cysβ-Hb 2 JCysβ-Ha,Cysβ-Hb 3 JGluR-H,Gluβ-Ha,b 3 JGluβ-Ha,Gluγ-Ha,b 2 JGluγ-Ha,Gluγ-Hb 3 JGluNH,GluR-H 3 JCysNH,CysR-H a
5.2 7.1 14.1 6.3 7.7 14.7 5.4
4.5 9.7 14.4 6.4 7.6 15.5 5.7a 7.6a
10.0 3.3 14.6 6.3 7.8 16.0 5.5 7.5
GSA major
GSA minor
17.2 6.7 8.5 14.8 8.7 6.7 12.0a 4.2a
17.1 5.4 10.2 14.8 5.5 9.5 12.0a 4.2a
At 2 °C.
(DSS,1 δ ) 0.015 ppm). The 13C chemical shifts (ppm) were referenced to internal 1,4-dioxane (dioxane, δ ) 66.0 ppm). The 15N chemical shifts (ppm) were referenced to external 15Nlabeled benzamide (δ ) 110.0 ppm). MS Studies. High-resolution electrospray ionization mass spectra (HRESIMS) were recorded on a Micromass LCT spectrometer using a probe voltage of 3200 V, an operating temperature of 150 °C, and a source temperature of 80 °C. Samples of 100 µM GSA were prepared in 50:50 acetonitrile/ water and infused into the instrument at a flow rate of 20 µL/ min. Leucine enkephalin was used as the lock mass internal standard. A mass (m/z) of 338.0656 was observed for GSA, which differs from the calculated mass of GSA by only -0.6 ppm. As a difference of
